Fig 1.
Identification and characterisation of lincRNA Ginir from mouse melanoma cells.
(A and B) The 5′ and 3′ ends of the 557-base Ginir transcript were extended using RACE. PCRs were performed with AP1 and AP2 and Ginir-specific primers G1F and G4R for 3′ RACE and primers G1R and G4F for 5′ RACE (A). For nested PCR, primary PCR products were used as templates. Primer sequences are enlisted in S4 Table included in the Materials and methods section. The 5′–3′ RACE was followed by Southern hybridisation with Ginir sequence–specific probes (B). (C) Schematic representation of Ginir (NCBI Acc. No: EF649772.1) on mouse chromosome X (ChrX: 61982243–61982854) was acquired using the UCSC genome browser (http://genome.ucsc.edu/cgi-bin/hgBlat) and GRCm38/mm10 Assembly. The bottom region shows ESTs and repeat elements spanning the Ginir locus. (D) The genomic region showing location of LDOC1 and MAGE genes spanning the contig bearing Ginir (www.ensembl.org). (E) Schematic representation of Ginir genomic sequence homology between mouse and rat species acquired using ENSEMBL (www.ensembl.org). AP1, adapter primer 1; AP2, adapter primer 2; EST, expressed sequence tag; Ginir, Genomic Instability Inducing RNA; LDOC1, LDOC1, Leucine Zipper down-regulated in cancer-1; lincRNA, long intergenic noncoding RNA; MAGE, the melanoma antigen gene; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; UCSC, University of California, Santa Cruz.
Fig 2.
Expression of Ginir/Giniras transcripts during mouse embryonic development and in adult tissues.
(A) List of several ESTs spanning Ginir sequence and showing significant similarity to Ginir (http://blast.ncbi.nlm.nih.gov/Blast.cgi). (B) RPA of RNA isolated from NIH/3T3 cells using PCR-generated sense or antisense riboprobes specific to Ginir sequence. Yeast total RNA served as control for RNase A/T1 activity. (C and D) Expression of Ginir/Giniras RNA in different stages of development (dpc) of mouse embryos (C) and in tissues from different organs of adult mice (D) using strand-specific cDNA synthesis and RT-PCR with G1F-G1R primers. Gapdh served as internal loading control. (E) Whole-mount ISH using LNA probes for Ginir (FAM labelled, green) or Giniras (TexRed labelled, red) on 10.5 dpc mouse embryos showing differential expression in brain (‘br’), forelimbs (‘fl’), and spinal cord (‘sc’). Whole-mount embryos treated with RNase A served as negative control for fluorescence. (F) FISH using LNA probes for Ginir (Green) or Giniras (Red) on embryo sections of 13.5 and 14.5 dpc embryos showing differential expression in forebrain (‘fb’), midbrain (‘mb’), hypothalamus (‘ht’), and limbs (‘li’). Embryo sections treated with RNase A served as a negative control. dpc, days post coitum; EST, expressed sequence tag; FAM, fluorescein amidite; FISH, fluorescence in situ hybridisation; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; ISH, in situ hybridisation; LNA, locked nucleic acid; PCR, polymerase chain reaction; RPA, ribonuclease protection assay; RT-PCR, reverse transcription PCR.
Fig 3.
Ectopic expression of Ginir RNA in cells leads to malignant transformation.
(A) Phase contrast micrographs of NIH/3T3-Ginir (three independent transfectants A, B, and C), NIH/3T3-EV, NIH/3T3-Giniras, and NIH/3T3-Ginir+Giniras cells. (Magnification 4×). (B) Cell proliferation analyses of NIH/3T3-Ginir (A, B, and C) individual transfectants, NIH/3T3-Giniras, and NIH/3T3-EV transfectant cells performed by MTT assay over a period of 6 days. Values are mean ± SEM (n = 3); **P ≤ 0.001 by repeated-measures one-way ANOVA test. (C) Cell proliferation analyses of NIH/3T3-EV, NIH/3T3-Ginir(A), NIH/3T3-Giniras, and NIH/3T3-Ginir+Giniras cells performed by MTT assay over a period of 7 days. Values are mean ± SEM (n = 3); ***P ≤ 0.0001; by repeated-measures one-way ANOVA test. (D) Representative xenograft tumours of NIH/3T3-Ginir(A) and NIH/3T3-Giniras cells generated in NOD/SCID mice. Tumour growth was monitored for 95 days. Tumourigenicity assays were performed at least 5 times with 3 mice in each group. (E) Tumour growth kinetics determined by measuring tumour volumes at an interval of 10 days over a period of 95 days. Values represent means ± SEM. ***P ≤ 0.0001, one tailed, by two-way ANOVA test. (F) Representative xenograft tumours of NIH/3T3-Ginir(B) and NIH/3T3-Ginir+Giniras cells generated in NOD/SCID mice. Tumour growth was monitored for 30 days (n = 3). Tumourigenicity assays were performed at least 3 times with 3 mice in each group. (G) Tumour growth kinetics monitored by measuring tumour volumes periodically up to duration of 30 days. Values represent mean ± SEM. ***P ≤ 0.0001, one tailed, by two-way ANOVA test. (H and I). In vivo lung colonisation assay in NOD/SCID mice performed via tail vein injection of NIH/3T3-Ginir(A) (H) and NIH/3T3-Ginir(B) (I) cells along with NIH/3T3-EV and NIH/3T3-Giniras cells as controls. Images represent lung tissues dissected from mice injected with mentioned cell lines after 6 (H) and 8 weeks (I) of introduction of cells. The assays were performed at least 3–5 times with n = 3 mice. (J and K) HE staining of lung tissue from mice injected with NIH/3T3-Ginir(A) (J) and NIH/3T3-Ginir(B) (K) cells. Supporting data for B, C, E and G can be found in S1 Data. Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA of Ginir; HE, haematoxylin–eosin; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.
Fig 4.
Down-regulation of Ginir RNA levels attenuates tumourigenic potential of NIH/3T3-Ginir cells.
(A) Representative RT-PCR analysis demonstrating Ginir levels in NIH/3T3-EV and NIH/3T3-Ginir(A) cells expressing Ginir-shRNA 1 and 2 and shControl (Scrambled) using G2F-G2R primers. Gapdh served as an internal RNA control. (B) Representative-phase contrast images of NIHGinir cells stably transfected with shRNAs to Ginir (shGinir1 or shGinir2) or with a scrambled shRNA (shControl). (Magnification 10×). (C) Cell proliferation analysis using MTT assay for the indicated cell lines. Data are mean ± SEM. ***P ≤ 0.0001, one tailed, paired Student’s t test. (D) Quantification of Ki67 antigen expression shown as percentage of positively stained cells compared to total number of cells per field (number of fields counted were at least 10) in indicated cell lines. Values are mean ± SEM. **P ≤ 0.001. Two tailed, by one-way ANOVA test. (E and F). Wound healing assays performed with NIH/3T3, NIHGinir-shControl, and NIHGinir-shGinir (1 and 2) cells. The wound gaps were observed at two different time points: 0 hours and 24 hours post wound formation (n = 3) (E). Statistical analysis of wound closure in mentioned cell types wherein the percentage of wound gap measured at indicated time points is plotted and the values are represented as mean ± SEM; ***P < 0.0001 by regular two-way ANOVA test (F). (G) Representative photographs of tumours demonstrating the inhibitory effects of Ginir-shRNA on tumour growth potential of NIH/3T3-Ginir(A) cells. Xenograft growth was monitored in NOD/SCID mice at regular intervals of 5 days for up to 40 days. Later, mice were killed, and tumours were dissected out. The assay was performed at least 3 times (n = 3). (H) Tumour growth kinetics of the mentioned cell lines monitored over a period of 40 days. Data are mean ± SEM; ***P value ≤ 0.0001 by regular two-way ANOVA test. Supporting data for C, D, F, and H can be found in S2 Data. Gapdh, glyceride 3-phosphate dehydrogenase; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; RT-PCR, reverse transcription polymerase chain reaction; shRNA, short hairpin RNA.
Fig 5.
Knock-down of endogenous Ginir affects proliferation and tumourigenic potential of fibroblasts and melanoma cells.
(A) Representative RT-PCR for analyses of Ginir knock-down in NIH/3T3 cells expressing Ginir-shRNA (1, 2, and scrambled control) using G2F-G2R primers. Gapdh served as an internal control. (B) Cell proliferation analysis by MTT assay for the indicated cell lines performed over a period of 4 days. Data presented are mean ± SEM. ***P ≤ 0.0001, one tailed, by two-way ANOVA test. (C) Quantification of Ki67 antigen–expressing cells shown as percentage of positively stained cells from the total number of cells present per field. Cells from at least 10 representative fields were counted in NIH/3T3 control and Ginir shRNA1 and 2 cells. Data presented are mean ± SEM.**P ≤ 0.001 by one-way ANOVA test (n = 3). (D) Quantitative analyses of cell cycle parameters of PI-stained cells in flow cytometry. Stable Ginir knock-down (NIHshGinir1, NIHshGinir2) cells were compared to NIH/3T3 control cells having endogenous Ginir RNA expression for percentage of cells in various phases of the cell cycle. Bars: means ± SEM; *P ≤ 0.05, **P ≤ 0.001; two tailed; by two-way ANOVA test. (E) Representative RT-PCR data demonstrating Ginir RNA expression levels in B16F10 melanoma cells shControl and the knock-down cells B16F10-Ginir-shRNA1 and B16F10-GinirshRNA2. PCR was done using G2F-G2R primers. Gapdh RNA expression was used for normalisation. (F) Phase contrast images of B16F10 control cells and cells stably transfected with shGinir1 or shGinir2. Arrowheads point towards cells showing altered phenotypes. Magnification 10×. (G) Cell proliferation analysis of indicated cells performed by MTT assay over a period of 4 days. Data shown are mean ± SEM. *P ≤ 0.05, one tailed, Student’s paired t test (n = 3). (H) Quantification of Ki67 antigen expression shown as percentage of positively stained cells as compared to the total number of cells per field. At least 10 fields were counted. Data shown are mean ± SEM. **P ≤ 0.001 by one-way ANOVA test. (n = 3). (I) Cell migration assay in the indicated cell lines. The gaps were measured after 6 hours using ImageJ software; version 1.41. Experiment was repeated at least thrice. (J) Quantitative analysis of relative wound recovery of each B16F10 Ginir knock-down cell induced by shRNA1 and shRNA2 as compared to B16F10-shGinir scrambled expressing control cells. Data represent mean ± SEM (n = 3). ***P ≤ 0.0001 by two-way ANOVA test. (K) Representative xenograft tumours of B16F10-shControl and B16F10-shGinir1 and B16F10shGinir2 (1 and 2) cells introduced into NOD/SCID mice; tumour growth was monitored for 15 days. These experiments were performed thrice, with 3 mice in each group. (L) Tumour growth kinetics were determined by measuring tumour volume each day for a period of 15 days for the indicated cell lines. Data shown represent mean ± SEM. ***P ≤ 0.0001, one tailed, one-way ANOVA test. Supporting data for B, C, D, G, H, and L can be found in S3 Data. Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PCR, polymerase chain reaction; PI, propidium iodide; RT-PCR, reverse transcription PCR; shRNA, short hairpin RNA.
Fig 6.
Ginir RNA is localised to cytoplasm in normal cells, but partitioning switches to the nuclear compartments in transformed cells.
(A-D) Subcellular fractionation followed by RT-PCR using G1F-G1R primers to determine compartmentalisation of Ginir and Giniras RNAs in (A) Clone M3, (B) NIH/3T3-EV, (C) NIH/3T3-Ginir, and (D) NIH/3T3-Giniras cells. Gapdh RNA served as an internal control for cytoplasmic RNA fractions and U6 snRNA as control for the nuclear fractions. The 18S rRNA served as a loading control for RT-PCR. A total of 35 cycles of amplifications were used for Ginir and Giniras RNAs, and 30 cycles were used for Gapdh RNA, U6 snRNA, and 18S rRNA. (E-H) RNA-FISH for Ginir localisation in the indicated cell lines using two independent LNA Ginir probes (Probe1-FAM labelled [E, G] and Probe2-Texas Red labelled [F, H]). Magnified images of a few cells are represented in G and H. Blue colour indicates nuclear staining with DAPI. Scale bar 10 μm. Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA to Ginir; LNA, locked nucleic acid; RNA-FISH, RNA fluorescence in situ hybridisation; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Fig 7.
Ginir RNA induces defects in DDR and increases DNA double-stranded breaks.
(A) Representative blot for expression of γH2AX, a DNA damage marker protein in NIH/3T3, NIH/3T3-Ginir (stable cell-line), and NIH/3T3-Ginir cells transiently transfected with Ginir cDNA (48 hours). Thirty μg of whole-cell protein lysates prepared from the mentioned cell lines was loaded on 16% SDS-PAGE. Gapdh protein served as an internal loading control. Data shown are representative of 3 independent experiments done with 3 independent transient and stable Ginir transfectants. (B) Representative images showing γH2AX expression in NIH/3T3 and NIH/3T3-Ginir (48 hours post transfection) cells. Blue colour indicates nuclear staining with DAPI. Scale bars 10 μm. (C) Quantification of γH2AX foci in mentioned cell lines wherein the percentage of cells demonstrating a given range of foci is plotted. The number of foci per cell were counted using the software tool Image J; version 1.41. At least 75 cells from 10 random fields were counted from both NIH/3T3 and NIH/3T3-Ginir cells (n = 5). (D) Representative western blotting for γH2AX protein in indicated cells. Thirty μg of whole-cell protein lysates was run on a 16% SDS PAGE and further processed for detection by chemiluminescence. Gapdh protein served as a loading control. (E) Images showing γH2AX expression in NIHGinir-shGinir scrambled and NIHGinir-shGinir 1 and NIH-Ginir shGinir2 cells. Blue colour indicates nuclear staining with DAPI. Scale bars 20 μm. (F) Quantification of γH2AX foci in mentioned cell lines using Image J tool; the number of foci per cell was counted for each cell type, and the percentage of cells showing specific number of foci is plotted. At least 100 cells from 10 random fields were counted for the mentioned cells. Estimation of foci number in each cell line was done at least 5 times. (G) Confocal images showing the activation of DDR proteins in NIH/3T3-EV and NIH/3T3-Ginir cells. Nuclei were stained with DAPI (blue). Scale bars 10 μm. (H) NIH/3T3 and NIH/3T3-Ginir cells analysed for DNA damage using Comet assay. (I) Brca1 protein expression in cells of NIH/3T3, NIH/3T3-Ginir in three independent transfectants A, B, and C. Fifty μg of whole-cell protein of mentioned cell lines was analysed on 7% SDS-PAGE. Gapdh protein served as an internal loading control. Supporting data for C and F can be found in S4 Data. ATM/ATR-S, ataxia–telangiectasia-mutated kinase and ataxia–telangiectasia and Rad3-related kinase substrate; Brca1, breast cancer type 1 susceptibility protein; DDR, DNA damage repair; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Mre11,meiotic recombination 11; pATM, ataxia–telangiectasia-mutated kinase.
Fig 8.
Ginir induces mitotic defects and impairs cytokinesis, forming multinucleated giant cells.
(A) Phase contrast micrographs demonstrating multinucleated giant cells present in mentioned cell lines. Encircled areas highlight the multinucleated giant cells amongst mononucleated proliferating cells (10× magnification). (B) Quantification of multinucleated cells in the indicated cell lines, expressed as percentage of cells with >2 nuclei. Nuclei were stained with DAPI. Images were acquired using confocal microscope. About 400 cells were counted manually for all cell lines except for NIH/3T3-Giniras, for which only about 200 cells were counted. Bars represent mean values ± SEM; **P values > .001 using Student’s t test. (C) Representative Kif20b staining in NIH/3T3-Ginir cells demonstrating the presence of multinucleated giant cells. Nuclei were stained with DAPI (blue) and viewed under confocal microscope. Scale bar 20 μm. (D and E) Representative RNA-FISH images stained with Ginir-specific LNA probe-1 (green) in NIH/3T3-Ginir cells (D). Magnified image of one of the multinucleated cells in NIH/3T3-Ginir cells. Nuclei were stained using DAPI (E). Scale bars 10 μm. (F) Representative Ki67 antigen staining in multinucleated giant cells of NIH/3T3-Ginir cells, demonstrating its strong expression. Nuclei were stained with DAPI. Scale bar 20 μm. (G) Immunofluorescence staining of the centrosome marker protein Ark1 in multinucleated giant cells. Nuclei were stained with DAPI. Scale bars 20 μm. (H) Immunofluorescence detection of the centrosome marker protein ɣ-tubulin in indicated interphase cells. Nuclei were stained with DAPI. Scale bars 5 and 10 μm. (I) Immunofluorescence detection of the centrosome marker protein Ark1 in mentioned interphase cells. Nuclei were stained with DAPI. Scale bars 5 μm. (J) Histogram plot demonstrating centrosome numbers in indicated cell lines. The percentage of cells with amplified (i.e., more than 2) and cells with a normal number of centrosomes (1 to 2) were visually scored from both metaphase and interphase cells. A minimum of 150 cells were counted for centrosome number by staining with γ-tubulin. The scoring was based on data from at least 3 independent experiments. Data shown represent mean ± SEM. ***P ≤ 0.0001, one tailed, one-way ANOVA test. (K) Images representing coexpression of a centrosome marker protein γ-tubulin with Ark1 in mitotically active cells. Nuclei were stained with DAPI. Scale bars 10 μm. Supporting data for B and J can be found in S5 Data. Ark1, aurora-related kinase 1; Ginir, Genomic Instability Inducing RNA; Kif20b, kinesin family member 20; RNA-FISH, RNA fluorescence in situ hybridisation.
Fig 9.
Ginir physically interacts with centrosomal protein Cep112 in cells.
(A) Schematic representation of RNA pull-down assay strategy using biotinylated Ginir RNA. (B) Western blot analysis for Cep112 protein expression. Fifty μg of whole-cell protein lysates of mentioned cell lines were loaded on 10% SDS-PAGE and probed using Cep112 antibody. (sc-246162, Santa Cruz). Tubulin protein served as internal loading control. (C) Western blot of Ginir RNA–associated proteins identified by RNA pull-down assays. NIH/3T3 cell proteins and 5′-biotinylated Ginir and Giniras RNAs were used in the assay. Elutes from RNA–protein complexes were loaded on 7% SDS-PAGE for protein separation. Blot was probed with Cep112 antibody (24928-1-AP, Proteintech). Biotinylated Giniras probe served as negative control. Unrelated biotin-labelled RNA probes like XEF RNA and Hotair RNA were used as nonspecific controls. Blot was stripped and reprobed for tubulin as control to rule out possibility of any nonspecific binding. (D) RIP with Cep112 antibody by using cell lysate of NIH/3T3-Ginir cells. RIP was followed by RT-PCR with Ginir RNA–specific primers (G2F-G2R). RT-PCR with Gapdh-specific primers served as control for nonspecific amplification in RIP. Also, RIP using anti-IgG and anti-Gapdh antibodies served as negative control for nonspecific binding. (E) RIP detection with Cep112 antibody for proteins isolated from NIH/3T3-Ginir cells followed by RT-PCR using primers that amplify Malat1 and U6 snRNA. These served as controls for nonspecific binding. (F) RIP assay with Cep112 antibody for analyses of interacting proteins sourced from NIH/3T3 and NIH/3T3-Ginir cells. Strand-specific RT-PCR was done using Ginir- and Giniras-specific primers (G2F-G2R). RIP with anti-IgG served as negative control for nonspecific binding. (G) Colocalisation of Cep112 with centrosomal marker γ-tubulin in mentioned interphase cells. Nuclear staining was done using DAPI. Scale bars, 10 μm. Cep112, centrosomal protein 112; Gapdh, glyceride 3-phosphate dehydrogenase; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA to Ginir; Hotair, HOX transcript antisense RNA; Malat1, metastasis-associated lung adenocarcinoma transcript 1; RIP, RNA-immunoprecipitation; MALDI-TOF, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA; XEF, Xenopus elongation factor.
Fig 10.
Ginir RNA impairs interaction between Cep112 and Brca1 proteins leading to genomic instability.
(A) RNA pull-down with biotinylated Ginir RNA in NIH/3T3 cells followed by western blotting with Brca1 antibody (sc-646, Santa Cruz). Pull-down with unbiotinylated RNA probe served as control for nonspecific binding. (B-D) RIP performed using both Cep112 and Brca1 antibodies followed by RNA isolation and RT-PCR with Ginir specific primers (G2F-G2R) in NIH/3T3-GinirA (B) and NIH/3T3-GinirB (C) cells. RIP assay was also followed by RT-PCR using nonspecific primers for U6 snRNA (D). Anti-IgG IP served as control for nonspecific interaction. (E) In silico model of Cep112 and Brca1 interaction generated through computational docking using ZDOCK tool. (F and G) Co-IP of Cep112 and Brca1 proteins in NIH/3T3-EV cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (sc-246162, Santa Cruz) (F) and vice versa (G). Anti-IgG IP served as a control for nonspecific binding to the antibody. (H and I) Western blotting with Cep112 antibody (24928-1-AP, Proteintech) for validation of Flag-Cep112 overexpression in NIH/3T3 cells (H). Fifty μg of whole-cell protein lysates from each of the mentioned cell lines was loaded on 7% SDS-PAGE. Tubulin served as internal loading control (I). (J) Co-IP of Brca1 and Flag-Cep112 in NIH-Flag-Cep112 cells. IP was performed with Flag1 antibody followed by immunoblotting with Brca1 antibody (20649-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (K) Co-IP of Brca1 and Cep112 proteins in NIH/3T3-EV, NIH/3T3-Ginir, and NIH-Ginir-shGinir2 cells. IP was performed with Brca1 antibody (sc-646, Santa Cruz) followed by immunoblotting with Cep112 antibody (24928-1-AP, Proteintech). Anti-IgG IP served as a control for nonspecific binding to the antibody. (L) Confocal images showing colocalisation of Brca1 with γ-tubulin in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 10 μm. (M) Confocal images showing colocalisation of Brca1 protein with Cep112 protein in NIH/3T3-EV cell line. Nuclei were stained with DAPI. Scale bars, 20 μm. (N) Confocal imaging for Brca1 expression in NIH/3T3-EV and NIH/3T3-Ginir cells. Scale bars, 20 μm. (O) RNA-FISH using Ginir-specific probe (probe 1, FAM labelled) in NIH/3T3-EV cells visualised by confocal imaging. Scale bars, 20 μm. (P) Co-IP of Brca1 and Cep112 in NIH/3T3-Ginir(C) cells wherein lysates were treated independently with RNase (A, H, and III mix) or RNasin. Both RNase- and RNasin-treated lysates were immunoprecipitated with Brca1 antibody (sc-646, Santa Cruz) and blotted using Cep112 antibody (sc-246163, Santa Cruz). IP with anti-IgG served as control. Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; FAM, fluorescein amidite; Ginir, Genomic Instability Inducing RNA; IgG, immunoglobulin G; IP, immunoprecipitation; RIP, RNA-immunoprecipitation; RNasin, RNase inhibitor; RT-PCR, reverse transcription polymerase chain reaction; snRNA, small nuclear RNA.
Fig 11.
Down-regulation of Brca1 and Cep112 proteins leads to increased genomic instability in NIH/3T3 cells.
(A and B) Representative immunoblots for Brca1 and Cep112 (sc-246163, Santa Cruz) proteins in NIH-siControl, NIH-siCep112, and NIH-siBrca1 cells. Cells were transfected with respective independent siRNA pools—siRNAs pool # 1 (Santa Cruz) (A) and siRNAs pool # 2 (Stealth RNAi, Thermo Fisher Scientific) (B). Fifty μg of whole-cell protein lysates of mentioned cell lines were loaded on 10% (A) and 7% (B) SDS-PAGE. Gapdh (A) and tubulin (B) proteins served as loading controls for the western blot. (C) Representative blot for γH2AX expression of NIH/3T3 cells transfected independently with Cep112 and Brca1 siRNA (pool # 1). Fifty μg of whole-cell protein lysates of each transfectant cell line was loaded on 16% SDS-PAGE. Gapdh protein served as loading control. (D-G) Confocal microscopy for detecting γH2AX foci along with nuclear staining with DAPI in siRNA pool # 1 (D) and siRNA pool # 2 (F) transfected NIH/3T3 cells. Scale bars, 10 μm. Quantification of γH2Ax foci in pool # 1 (E) and pool # 2 (G) cell lines using Image J tool. The number of foci per cell was counted in >75 cells (E) and >150 cells (G). The percentage of cells showing a given number of foci is plotted. Data shown represent mean ± SEM. ***P ≤ 0.0001, one tailed, one-way ANOVA test. (n = 3). (H and I) Immunofluorescence staining with Ark1 (H) and γ-tubulin (I) antibody in NIH-siControl, NIH-siCep112, and NIH-siBrca1 (pool # 1[H], pool # 2[I]) cells. Nuclei were stained with DAPI. Scale bars, 20 μm (H), 10 μm (I). (J) Histogram plot demonstrating centrosome numbers in represented cell lines. The percentage of cells with normal number of centrosomes (1 to 2) or cells with amplified (i.e., more than two centrosomes) were visually scored in both metaphase and interphase cells. The scoring was done based on γ-tubulin staining examined in at least 150 cells. A minimum of three independent experiments were done for each cell line. Data shown represent mean ± SEM. ***P ≤ 0.0001, one tailed, one-way ANOVA test. Supporting data for Figs E, G, and J can be found in S6 Data. Ark1, aurora-related kinase 1; Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; Gapdh, glyceride 3-phosphate dehydrogenase; siRNA, small interfering RNA.
Fig 12.
Model proposing mechanism through which Ginir mediates malignant transformation in mouse cells.
Brca1, breast cancer type 1 susceptibility protein; Cep112, centrosomal protein 112; DDR, DNA damage response; Ginir, Genomic Instability Inducing RNA; Giniras, antisense RNA to Ginir.